Standardisation of sequencing coverage depth in NGS: recommendation for detection of clonal and subclonal mutations in cancer diagnostics

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00098892%3A_____%2F19%3AN0000032" target="_blank" >RIV/00098892:_____/19:N0000032 - isvavai.cz</a>

Alternative codes found

RIV/61989592:15110/19:73594809

Result on the web

<a href="https://www.frontiersin.org/articles/10.3389/fonc.2019.00851/full" target="_blank" >https://www.frontiersin.org/articles/10.3389/fonc.2019.00851/full</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.3389/fonc.2019.00851" target="_blank" >10.3389/fonc.2019.00851</a>

Result language

angličtina

Original language name

Standardisation of sequencing coverage depth in NGS: recommendation for detection of clonal and subclonal mutations in cancer diagnostics

Original language description

The insufficient standardization of diagnostic next-generation sequencing (NGS) still limits its implementation in clinical practice, with the correct detection of mutations at low variant allele frequencies (VAF) facing particular challenges. We address here the standardization of sequencing coverage depth in order to minimize the probability of false positive and false negative results, the latter being underestimated in clinical NGS. There is currently no consensus on the minimum coverage depth, and so each laboratory has to set its own parameters. To assist laboratories with the determination of the minimum coverage parameters, we provide here a user-friendly coverage calculator. Using the sequencing error only, we recommend a minimum depth of coverage of 1,650 together with a threshold of at least 30mutated reads for a targeted NGSmutation analysis of≥3% VAF, based on the binomial probability distribution. Moreover, our calculator also allows adding assay-specific errors occurring during DNA processing and library preparation, thus calculating with an overall error of a specific NGS assay. The estimation of correct coverage depth is recommended as a starting point when assessing thresholds of NGS assay. Our study also points to the need for guidance regarding the minimum technical requirements, which based on our experience should include the limit of detection (LOD), overall NGS assay error, input, source and quality of DNA, coverage depth, number of variant supporting reads, and total number of target reads covering variant region. Further studies are needed to define the minimum technical requirements and its reporting in diagnostic NGS.

Czech name

—

Czech description

—

Type

J_imp - Article in a specialist periodical, which is included in the Web of Science database

CEP classification

—

OECD FORD branch

30205 - Hematology

Project

<a href="/en/project/NV16-32339A" target="_blank" >NV16-32339A: Impact of functional polymorphisms influencing inflammation and oxidative stress on outcome and selection of treatment in chronic lymphocytic leukemia</a><br>

Continuities

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)<br>I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Publication year

2019

Confidentiality

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Name of the periodical

Frontiers in Oncology

ISSN

2234-943X

e-ISSN

2234-943X

Volume of the periodical

9

Issue of the periodical within the volume

September 2019

Country of publishing house

CH - SWITZERLAND

Number of pages

6

Pages from-to

851

UT code for WoS article

000483735300001

EID of the result in the Scopus database

2-s2.0-85073060022